Immunization with attenuated sporozoites (gamma-irradiated or genetically modified) induces sterile immunity that prevents malaria infection. Attenuated sporozoites maintain their ability to infect the hepatocyte but arrest at an early stage of intrahepatocytic development. To develop a sub-unit vaccine based on this model, we need to identify the targets of immunity. Previous studies in cancer research demonstrated that heat shock protein (HSP)-chaperoned peptides derived from tumor cells can induce immune responses against the same tumor. To identify peptides carried by HSPs for presentation to the immune system the following experimental approaches are used: 1. HSP-peptide complexes are purified from infected hepatocytes followed by peptide elution and identification by mass spectrometry 2. In-vitro binding studies: purified HSP from uninfected hepatocyte are incubated with a peptide mixture obtained from sporozoite. Bound peptides are eluted from the HSP and analyzed by mass spectrometry. The corresponding proteins will be used to identify orthologs expressed by human parasites. Protein that uniquely expressed during the sporozoite-liver stage developmental stages of the parasites and have orthologs expressed by human parasites will be used to immunize mice followed by challenge with wild type sporozoites to identify protective HSP-peptide complexes. Published methods for purifying HSPs from tumor cells were adapted to purify HSPs from mice hepatocytes, resulting in optimized protocols to obtain several of the main HSPs (HSP70, HSP90 and gp96) in a pure form. Preliminary results from in-vitro binding assay of sporozoite peptides to gp96 identified peptides corresponding to 50 malaria proteins that bound to gp96. Of those, 11 peptides derived from 10 proteins exclusively expressed by sporozoite/liver stage parasites were eluted from gp96 but were not identified in the unbound fraction. We will confirm these finding in repeated assays and in in-vivo studies of purified HSP-peptide complexes from malaria-infected mice.